The fibroblast growth factor (FGF) family comprises over twenty structurally related proteins. These proteins exert a wide range of effects on cells of the vascular, neural, endocrine, and immune systems. That is, the FGF family proteins can induce such events as angiogenesis, regeneration, and morphogenesis. For instance, members of the FGF family have been implicated in such processes as, but not limited to, cell migration, proliferation, and differentiation (Burgess and Maciag 1989, Ann. Rev. Biochem. 58:575; Thomas, 1987, FASEB J. 1:434; Baird and Bohlen, 1990, In: Peptide Growth Factors and their Receptors, pp. 369–418, Sporn et al., eds., Springer-Verlag, New York; Bjornsson et al., 1991, Proc. Natl. Acad. Sci. USA 88:8651).
Moreover, members of the FGF family are potent regulators of developmental, physiological and pathophysiologic events in mammals. Therefore, these are important molecules in the development of therapeutics relating to these events.
The most well-studied members of the FGF family are FGF-1 and FGF-2, which have been referred to under various names, most often as acidic FGF and basic FGF, respectively. Both factors have been characterized extensively as described, for instance, in U.S. Pat. No. 5,849,538, U.S. Pat. No. 5,827,826, U.S. Pat. No. 5,571,790, U.S. Pat. No. 5,552,528, and U.S. Pat. No. 4,868,113.
Although there is a large overlap in the spectrum of receptor-binding properties and biological activities shared among the FGFs, the only known function shared by all members of the family is a relatively high affinity for heparin or heparan sulfate. It has also been demonstrated that binding with heparin can potentiate the mitogenic activity of FGF-1 and can protect both FGF-1 and FGF-2 from inactivation by proteolysis and heat.
As peptide growth factors, the mitogenic activities of FGFs are mediated by a high affinity receptor located at the plasma membrane. Numerous reports have shown that FGF-1 has potent angiogenic activity and nerve regeneration inducing ability. Angiogenesis and nerve regeneration are two events that must occur following injury to restore the tissue to a normal functional state following injury. Thus, FGF-1 has the potential to be used as a therapeutic agent to promote such events as angiogenesis and nerve regeneration. For example, FGF-1 is a good candidate to promote angiogenesis in the heart following myocardial ischemia.
Despite the important role of FGF-1 in mediating repair of tissue injury, studies where FGF-1 was administered by intravenous and intracoronary techniques to induce angiogenesis in an injured heart were not particularly successful. This failure was primarily due to insufficient amounts of biologically active FGF-1 reaching the designated target area. Without wishing to be bound by any particular theory, these failures have been thought to be due in part to the proteolytic degradation of FGF-1 before it can reach the ischemic area. Thus, a more protease resistant, or degradation resistant, form of FGF-1 would significantly enhance the therapeutic potential of FGF-1.
More specifically, therapeutic use of native or wild-type FGF-1 (wt FGF-1) has been impeded because it is susceptible to cleavage by thrombin (Lobb, 1988, Biochemistry 27:2572). This is particularly important in wound healing, repair, and angiogenesis because of the intimate anatomical relationship between nerves and vasculature such that tissue injury results in concurrent damage to both the neural and vascular systems and the damaged area is generally embedded in a fibrin clot. Because much of the thrombin generated during coagulation associates with fibrin, high concentrations of thrombin are typically present at the site of injury. Under such conditions, FGF-1 is rapidly cleaved to smaller biologically less active and/or inactive fragments due to thrombin degradation. Thus, thrombin degradation inhibits the therapeutic effects of FGF-1 at the site of injury.
To date, prior art methods for administration of FGF-1 to a site of interest have not considered protecting it from thrombin degradation and have been unsuccessful. In addition, a potential way of delivering FGF-1 locally to the site of injury involves its incorporation into fibrin sealant. Fibrin sealant, as disclosed in, e.g., International Publication No. WO 92/09301, and WO 94/20133, and U.S. Pat. No. 6,117,425, can comprise a fibrinogen solution comprising plasma components such as, but not limited to, fibrinogen and factor XIII, as well as thrombin to which calcium ions are added. Equal volumes of fibrinogen and thrombin solutions are mixed together prior to use and a resulting fibrin polymer seal forms. Thrombin catalyzes the release of fibrinopeptides A and B from fibrinogen to produce fibrin monomers, which aggregate to form fibrin filaments. Thrombin also activates the transglutaminase, factor XIIIa, which catalyzes the formation of isopeptide bonds to covalently cross-link the fibrin filaments. This procedure can be modified to be used as a prolong delivery device. Growth factors, which bind to fibrin, are added to the fibrinogen solution. The resulting fibrin polymer can therefore comprise bound growth factor and, as the fibrin is degraded by naturally occurring enzymes, the growth factors would be released into the surrounding area. More specifically, WO 92/09301, WO 94/20133, and U.S. Pat. No. 6,117,425, demonstrate that FGF-1 in the presence or absence of heparin, can be mixed with fibrinogen before the addition of thrombin. Thrombin cleaved fibrinogen into fibrin thereby producing a fibrin matrix containing the FGF-1 and heparin. As the body's fibrinolytic system slowly degraded the fibrin matrix, the growth factor and heparin were expected to be released by this system.
Unfortunately, the addition of thrombin to the system degrades the FGF-1, requiring high concentrations of FGF-1 to be added in order to ensure that enough intact, active FGF-1 remained available to be released at the target site. Therefore, the fibrin sealant system, while potentially useful for sustained delivery of other growth factors, which are not susceptible to thrombin degradation, is currently not optimized for delivery of FGF-1. Thus, while a thrombin-resistant form of FGF-1 could be administered via the fibrin sealant system, such an FGF-1 is not available.
In conclusion, there is a long-felt need for methods of delivering active FGF-1 to a site of interest thereby promoting processes such as angiogenesis, nerve regeneration, and wound repair, among others. Further, there is a long-standing need for FGF-1 resistant to thrombin degradation. The present invention meets these needs.